14-9966-95

antibody from Invitrogen Antibodies

Targeting: GATA3 HDR

Provider product page for 14-9966-95

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Antibody data

Antibody Data
Antigen structure
References [55]
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Validations
Western blot [1]
Immunocytochemistry [2]
Other assay [22]

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Product number: 14-9966-95 - Provider product page
Provider: Invitrogen Antibodies
Product name: Gata-3 Monoclonal Antibody (TWAJ), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The monoclonal antibody TWAJ recognizes mouse and human Gata-3, a member of the Gata family of transcription factors. Gata-3 is a T cell-specific transcription factor important for thymic development and Th2 differentiation. Expression during embryonic development is found in the central nervous system, skin, mammary glands and kidney. During development, the expression of Gata-3 is essential as homozygous knock-out of Gata-3 is embryonic lethal. The Gata-3 is also essential for T cell commitment and survival. In the thymus, expression is found mainly on the CD4 single positive cells. During Th2 differentiation, Gata-3 binds to the IL-4 promoter as well as represses the expression of T-bet, thus inhibiting Th1 differentiation.
Antibody clone number: TWAJ
Concentration: 0.5 mg/mL

GATA3 protein structure - 14-9966-95 shown in red.

Supportive validation

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Experimental details: Immunofluorescence analysis of GATA-3 was performed using 70% confluent log phase MCF7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with GATA-3 Monoclonal Antibody (TWAJ) (Product # 14-9966-80) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight, followed by labelling with Goat anti-Rat IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A11006) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

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Experimental details: Knockdown of GATA-3 was achieved by transfecting MCF7 cells with GATA-3 specific siRNAs (Silencer® select Product # s5600; s5599). Immunofluorescence analysis was performed using untransfected MCF7 (panels a, d), transfected with non-specific scrambled siRNA (panels b,e) and transfected with GATA-3 specific siRNAs (panel c,f). The cells were labeled with GATA-3 Monoclonal Antibody (TWAJ) (Product # 14-9966-80) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight, followed by labelling with Goat anti-Rat IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A11006) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962) and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Reduction of GATA-3 expression was observed upon siRNA mediated knockdown (panel c,f), confirming the specificity of the antibody to GATA-3. The images were captured at 60X magnification.

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Experimental details: 7 Inhibition of CK2 activity modulates mRNA expression of IRFs in MoDCs. Immature MoDCs were pretreated for 1 h with DMSO 0.1% or CX-4945 (CX, 5 uM) and then stimulated for 6 h with (A) NiSO 4 500 uM or (B) DNCB 10 uM. IRF-1, -8, and -4 mRNAs were quantified by real-time RT-PCR. Naive CD4 + T lymphocytes were coincubated with NiSO4-stimulated MoDCs (C) in the absence or presence of CX-4945 (5 uM). On day 5, cells were restimulated with anti-CD3/CD2/CD28 beads for 48 h and stained with CD4-Alexa Fluor 700, CD3-FITC, RorgammaT-APC, GATA3-PE, and T-Bet Brilliant Violet 605. Living cells (ZombieAqua negative cells) were analyzed by flow cytometry. Data represent the results of 5 independent experiments and are normalized to the control. * P < 0.05; ** P < 0.01 (Mann-Whitney test).

Supportive validation

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Experimental details: Fig 5 Lentiviral-mmu-miR-135a treatment influences Th cell polarization. The expression of T-bet and GATA-3 protein in CD4 + T cells was measured in the spleens of normal (control), AR (AR-induced), positive (AR-induced, treated with lentiviral-mmu-miR-135a), and negative (AR-induced, treated with empty lentivirus) mice using flow cytometry. (A) Representative dot plots from each experimental group. The percentages of CD4 + T-bet + T cells (B) and CD4 + GATA-3 + T cells (C) were also calculated. Data are presented as the mean +- SEM. *P

Supportive validation

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Experimental details: Fig. 7 Tumor burden is reduced in St2 receptor-deficient gp130 FF mice. a Quantification of total tumor burden in 100-day-old mice of the indicated genotype. Each symbol represents an individual mouse. One-way ANOVA was performed with F (DFn, Dfd) = 11.83 (2, 48). b Enumeration of total tumor number from mice in a , and of tumors following classification according to their size. n = 12 ( FF, St2 +/+ ), n = 20 ( FF, St2 +/ - ), and n = 19 ( FF, St2 -/- ) mice. One-way ANOVA was performed with F (DFn, Dfd) = 22.79 (11, 192). c Quantification of toluidine blue (for detection of mast cells; submucosal tissue), F4/80 and CD31 stained sections of gastric tumors of mice of the indicated genotype. Mast cells: n = 10 mice, t -test t (df) = 4.25 (18); F4/80: n = 8 (FF; St2 +/+ ), n = 9 (FF; St2 - /- ), one-way ANOVA F (DFn, Dfd) = 27.52 (3,29); CD31: n = 6 (Submucosa) n = 5 (Tumor), and one-way ANOVA F (DFn, Dfd) = 13.6 (3,19). d qPCR expression analysis of chemokines expressed by FACS-purified tumor-associated mast cells from stomachs of either FF; St2 +/+ or FF; St2 -/- mice. All n = 4 from two independent experiments. Csf2 : t -test t (df) = 3.81 (6); Ccl3 : t -test t (df) = 3.97 (6); Ccl7 : t -test t (df) = 0.88(6); Il6 :: t -test t (df) = 4.02 (6); e , f Flow cytometric analysis of unaffected antrum (AN) and antrum tumors (AT) of indicated genotype for the frequency of ILC2 cells (lineage - , Cd11b - , Gata3 + ), Tregs (Foxp3 + , CD4 + ), and proportion of St2 + cells within these

Supportive validation

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Experimental details: Fig. 6 Inactivation of Tfap2a and Tfap2b by CRISPR/Cas9 leads to defects in nephric duct morphogenesis and progenitor cell identity. a Schematic representation of Tfap2a and Tfap2b exons targeted by CRISPR/Cas9 technology and the location of the sgRNAs used. b Wholemount GFP fluorescence of control ( Pax2-GFP ) and allelic series of Tfap2a/2b ; Pax2-GFP mutant embryos at E9.5 and E10.5. White arrows denote nephric duct integrity defects whereas yellow arrow and inset magnification highlight ectopic Pax2-GFP positive cells. Scale bar of the inset = 50 mum. c Quantification of nephric duct (ND) elongation in Tfap2a/2b ; Pax2-GFP double KO embryos at E9.5. n = 15 (Control) and n = 4 ( Tfap2a/2b double KO) biologically independent samples. The graphs represent mean +- SD, assessed by a two-tailed Mann-Whitney test. Source data are provided as a Source Data file . d Immunostaining for the nephric duct marker Gata3 in transverse sections of E9.5 Tfap2a/2b double mutant shows an elongation defect and the presence of ectopic Gata3 positive (nephric duct) cells (yellow arrows). Nephric duct cells are denoted by white dotted lines. Scale bar 25 mum for all pictures. e Quantification of Gata3 + cells in the rostral region of E9.5 control and Tfap2a/2b double KO embryos ( n = 7). The graph represents mean +- SD, determined by a two-tailed unpaired t -test. Source data are provided as a Source Data file . f Immunostaining for the markers Hoxb9 (NdPr1), and intermediate mesoderm (Wt1) in t

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Experimental details: Figure 1 Homozygous RhoA deletion in Treg cells leads to early, fatal spontaneous inflammatory disorders. (A) Survival outcome of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice. Results were analyzed with a log-rank (Mantel-Cox) test and expressed as Kaplan-Meier survival curves. (B) Image of lymphadenopathy in RhoA Flox/Flox Foxp3 YFP-Cre mice. Inguinal lymph nodes are shown. (C) Images of H&E staining of the indicated organs from RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice (original magnification X 400). (D) Left, representative flow cytogram of CD44 and CD62L staining in CD4 + and CD8 + cells from the spleen of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice. The numbers indicate percentages of CD44 + , CD44 + CD62L + , and CD62L + cells. Right, average percentages of CD44 + , CD44 + CD62L + , and CD62L + cells. (E) Left, representative flow cytogram of IL-17, IFN-gamma, and IL-4 staining in CD4 + Foxp3 - cells from the spleen of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice. The numbers indicate percentages of IL-17 + , IFN-gamma + , and IL-4 + cells. Right, average percentages of IL-17 + , IFN-gamma + , and IL-4 + cells. (F) Left, representative flow cytogram of RORgammaT, T-bet and GATA3 staining in CD4 + Foxp3 - cells from the spleen of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice. The numbers indicate percentages of RORgammaT + , T-bet + , and GATA3 + cells. Right, average percentages of RORgam

Supportive validation

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Experimental details: Figure 2 Homozygous RhoA deletion in Treg cells dampens Treg cell homeostasis and induces Treg cell plasticity. (A) Left, representative flow cytogram of Foxp3 staining in CD4 + cells from the spleen of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/Flox Foxp3 YFP-Cre mice. The numbers indicate percentages of CD4 + Foxp3 + Treg cells. Middle, average percentages of CD4 + Foxp3 + Treg cells. Right, average numbers of CD4 + Foxp3 + Treg cells. (B) Treg cell proliferation. Percentages of CD4 + Foxp3 + Treg cells incorporated with BrdU are shown. (C) Treg cell apoptosis. The expression levels (MFI: Mean fluorescence intensity) of active caspase 3 in CD4 + Foxp3 + Treg cells are shown. (D) The expression levels of Foxp3 in Treg cells. (E) Left, representative flow cytogram of IL-17, IFN-gamma, and IL-4 staining in CD4 + Foxp3 + Treg cells. The numbers indicate percentages of IL-17 + , IFN-gamma + , and IL-4 + Treg cells. Right, average percentages of IL-17 + , IFN-gamma + , and IL-4 + Treg cells. (F) Left, representative flow cytogram of RORgammaT, T-bet and GATA3 staining in CD4 + Foxp3 + Treg cells. The numbers indicate percentages of RORgammaT + , T-bet + , and GATA3 + Treg cells. Right, average percentages of RORgammaT + , T-bet + , and GATA3 + Treg cells. (G) Left, representative histogram of the expression levels of CTLA-4, GITR and PD-1 in CD4 + Foxp3 + Treg cells. The numbers above the graphs indicate MFI. Right, average MFI of CTLA-4, GITR and PD-1 in CD4 + Foxp3 + Treg cells. n =

Supportive validation

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Experimental details: Figure 5 Heterozygous RhoA deletion in Treg cells induces Treg cell plasticity and increases CD4 + effector T cells but does not result in autoimmunity. (A) Body weight of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/+ Foxp3 YFP-Cre mice. (B) Images of H&E staining of the indicated organs. (C) Representative flow cytogram of Foxp3 staining in CD4 + cells from the spleen of RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/+ Foxp3 YFP-Cre mice. The numbers indicate percentages of CD4 + Foxp3 + Treg cells. (D) Left, average percentages of CD4 + Foxp3 + Treg cells. Right, cell numbers of CD4 + Foxp3 + Treg cells. (E) Proliferation of Foxp3 + YFP + Treg cells from RhoA +/+ Foxp3 YFP-Cre/+ and RhoA Flox/+ Foxp3 YFP-Cre/+ female mice. Percentages of Foxp3 + YFP + Treg cells incorporated with BrdU are shown. (F) Apoptosis of Foxp3 + YFP + Treg cells from RhoA +/+ Foxp3 YFP-Cre/+ and RhoA Flox/+ Foxp3 YFP-Cre/+ female mice. The expression levels (MFI: mean fluorescence intensity) of active caspase 3 in Foxp3 + YFP + Treg cells are shown. (G) The expression levels of Foxp3 in Treg cells from RhoA +/+ Foxp3 YFP-Cre and RhoA Flox/+ Foxp3 YFP-Cre mice. (H) Representative flow cytogram of IFN-gamma, IL-17 and IL-4 staining in CD4 + Foxp3 + Treg cells. The numbers indicate percentages of IFN-gamma + , IL-17 + and IL-4 + Treg cells. (I) Average percentages of IFN-gamma + , IL-17 + and IL-4 + Treg cells. (J) Representative flow cytogram of RORgammaT, T-bet and GATA3 staining in CD4 + Foxp3 + Treg cells. The numb

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Experimental details: Figure 2 Murine carcinogen-induced oral lesions are enriched for CD4 + Treg and conventional T cells 6- to 8-week-old C57BL/6 mice were exposed to drinking water containing 100 mug/mL 4-NQO or vehicle control for 20 weeks. (A) Quantification of histopathology. After 20 weeks of treatment with 4-NQO drinking water, tongues were dissected, fixed in 10% formalin, bisected longitudinally, and stained with H&E. The perimeter of each tongue is outlined and categorized base on histology grade: hyperkeratosis (black); dysplasia (blue); or SCC (green). Shown is a representative H&E stain of FFPE tongue after longitudinal bisection of 4-NQO-treated mouse, perimeter traced based on histology grades noted above. (B and C) Summary plots of 4-NQO-induced histopathology, showing the percentage of tongue perimeter defined as indicated histology grade. Each bar (B) or symbol (C) represents an individual tongue from a single mouse. Median is indicated in (C). n = 10 mice. (D) (Top) Representative H&E image of SCC region of tongue epithelium from (A). (Bottom) Representative CD3 (left) and Foxp3 (right) IHC images of adjacent sections of depicted SCC region denoted by white box in H&E image are shown. White arrows denote CD3 + or Foxp3 + cells. Scale bars represent 400 mum (H&E) or 50 mum (IHC). (E) Summary plot of pooled data from CD3 IHC density analysis, showing the number of CD3 + cells per mm 2 for each lesion. Each symbol represents an individual lesion. Median is indicated. n = 10 region

Supportive validation

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Experimental details: Figure 2. Histone deacetylase 3 (HDAC3)-deficient CD4 + T cells from dLck-Cre HDAC3 cKO mice have reduced differentiated T h -cell populations. Identification of helper T-cell populations in vivo. Splenocytes were harvested from wild-type (WT) and HDAC3 cKO mice, and labeled for flow cytometry. Cells were first gated on HDAC3 + or HDAC3 - events, then gating for T h 1 (T-bet + ), T h 2 (GATA3 + ), T h 17 (RORgammat + ), T reg (Foxp3 + CD25 + ), and T fh (CXCR5 + PD-1 + Bcl-6 hi ) is shown (left). Bar plots on right represent pooled data for the total cell number +- standard deviation (SD) from three independent experiments ( n = 4-5 mice/group in total). Non-T fh CXCR5 - PD-1 - cells (dark gray histograms) were used as a negative control for Bcl-6 expression to set the gate on the Bcl-6 histograms. Statistical significance was determined for the indicated comparisons using ordinary one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Figure 2--figure supplement 1. Histone deacetylase 3 (HDAC3)-deficient T cells from dLck-Cre HDAC3 cKO mice have reduced differentiated T h -cell populations. Identification of helper T-cell populations in vivo. Lymphocytes were harvested from mesenteric lymph nodes of wild-type (WT) and dLck-Cre HDAC3 cKO mice, and labeled for flow cytometry. Cells were first gated on HDAC3 + or HDAC3 - events, then gating for T h 1 (T-bet + ), T h 2 (GATA3 + ), T h 17 (RORgammat + ),

Supportive validation

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Experimental details: Extended Data Figure 7 Distinguishing the drivers of autoimmunity in the absence of T-bet + Treg cells a,b , ex-Treg cells are no more pathogenic than effector CD4 T cells. a , CD4 + CD25 + (Treg) cells were sorted from lymph nodes of Tbx21 RFP-Cre Foxp3 WT mice, and CD4 + CD25 - (effector) and CD4 + CD25 lo (exTreg) cells were sorted from lymph nodes of Tbx21 RFP-Cre Foxp3 FL mice for transfer into Tcrb - / - Tcrd - / - mice. Intracellular staining for Foxp3 demonstrates purity of cell populations. b , Weights of Tcrb - / - Tcrd - / - mice receiving CD4 + CD25 + (white squares), CD4 + CD25 - (black squares), and CD4 + CD25 lo (gray squares) cells. c , Percentages and numbers of the indicated T cell populations in spleens of mice analyzed on d62 post transfer. d,e , T-bet + effector alphabetaT cells drive disease upon ablation of T-bet + Treg cells. Lethally irradiated Tcrb - / - Tcrd - / - mice were reconstituted with a 1:1 mix of CD45.2 + Tbx21 RFP-Cre/WT R26 iDTR T-cell depleted bone marrow cells with either CD45.1 + Foxp3 KO , CD45.1 + Foxp3 WT , or CD45.2 + Tcrb KO T-cell depleted bone marrow cells. Mice were injected with 0.5mug diphtheria toxin (DT) on day 0, then treated daily with 0.1mug DT for 22 days before analysis. d , Weight loss in Tbx21 RFP-Cre/WT R26 iDTR :Foxp3 KO (red line) vs. Tbx21 RFP-Cre/WT R26 iDTR :Foxp3 WT (black line) vs. Tbx21 RFP-Cre/WT R26 iDTR :Tcrb KO (blue line) reconstituted mice. e , Representative fl

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Experimental details: Figure 2 OX40 Is Critical for Th2 and Treg Cell Response to IL-33 and Papain (A) TNFRSF gene expression was analyzed in the indicated immune cell populations using IMMGEN and naive lung ILC2 microarray data. (B and C) OX40 expression was analyzed on lung GATA3 + and GATA3 - Treg and Th2 cells on day 5 from WT mice treated with PBS, papain, or IL-33 (i.n., day 0 and 1). Fluorescence minus one (FMO) control was used to establish gating threshold. Percent OX40 + cells were calculated in the indicated populations and treatments and compared to Foxp3 - GATA3 - (DN) CD4 + T cells. (D-F) Lung GATA3 + and GATA3 - Treg and Th2 cells were quantified from WT and Tnfrsf4 -/- mice on day 5 after IL-33 treatment (i.n., day 0 and 1). (G-I) Lung GATA3 + and GATA3 - Treg and Th2 cells were quantified from WT and Tnfrsf4 -/- mice on day 5 after papain (Pap) treatment (i.n., day 0 and 1). Bar graphs indicate mean (+-SEM). (A), two independent datasets per group; (B) and (C), ANOVA, two repeat experiments; (D)-(F), ANOVA, two repeat experiments; (G)-(I), ANOVA, two repeat experiments. *** p

Supportive validation

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Experimental details: Figure 4. Duodenal infection leads to a compartmentalized immune conflict in the duodenal gLNs and to compromised oral tolerance. a , Dissected gLNs from non-infected (N.I.) C57BL/6 mice or mice infected with 700 S. venezuelensis (S.v.) larvae 8 days prior to harvest. b , Total CD45 + cell counts ( n =5, represents 3 independent experiments). c-f , Frequency of CD103 + CD11b - ( c ), CD103 + CD11b + ( d ) and CD103 - CD11b + ( e ) among MHCII hi CD11c + cells and eosinophils among CD45 + cells ( f ) ( n =5, represents 2 independent experiments). g , Representative flow cytometry plot of GATA3 + and FOXP3 + CD4 + T cells. h , Frequency of GATA3 + CD4 + T cells ( n =5, represents 3 independent experiments). i , Frequency of total FOXP3 + among CD45.1 + cells in gLNs 64 h post adoptive transfer of 1 x 10 6 naive CD45.1 + OT-II cells into CD45.2 mice ( n =8, pool of 2 independent experiments) infected with S.v. larvae or N.I. 8 days and gavaged with OVA 48 h and 24 h prior to analysis. j , Scheme of oral tolerance experimental set-up in S.v. infected mice. k - m , Total eosinophils in bronchoalveolar lavage fluid (BALF) ( k ), frequency of eosinophils among CD45 + cells in lung tissue ( l ) and OVA-specific IgG1 levels in serum ( m ) from mice infected with S.v. or N.I. during antigen feeding (+OVA groups) or no feeding (-OVA groups), at 21 d after first immunization with OVA-alum ( j ) ( n =13 for +OVA groups, n =10 for -OVA groups, pool of 2 independent experiments). LN abbrevi

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Experimental details: Figure 14 Long-lived memory CD4 + T cells, without uropodia, have a lower threshold for re-stimulation, when they make further, independent fate decisions to develop uropodia or re-express CD62L. (A-C) Cell trace violet (CTV) labeled female A1RAG CD4 + T cells were stimulated with bone marrow-derived dendritic cells (bmDC) + 100 nM Dby for 3 days in the presence of IL2 (50 U/ml) without TGFbeta (A) , IL2 (50 U/ml) plus TGFbeta (2 ng/ml) (B) , or TGFbeta plus anti-IL2 (clone S4B6, 50 ug/ml) (C) . Cells were "" in situ "" labeled with live/dead aqua, fixed and permeabilized, then 7AAD. Histograms show the absolute frequencies of CTV dilution, with gray dashed lines for all images (both live and dead), while filled red (cells with uropodia >10 mm 2 ) and blue line (cells without uropodia) histograms are gated for live cells only (live/dead aqua negative, bright field contrast low). Total numbers of cells in each histogram are indicated. One of two similar experiments is shown. (D-F) CTV labeled female MARKI CD4 + T cells were stimulated with bmDC + 10 nM Dby peptide with IL2 (50 U/ml) either with (E,F) or without (D) TGFbeta (2 ng/ml) for 10 days. Cells were "" in situ "" labeled with live/dead aqua and Mitotracker DR (not shown), fixed and permeabilized, then Tbet-Alexa488, foxp3-PE-Cy7, (GATA3-PE, RORgammat-PE-CF594, CD4-APC-Cy7, all not shown), and 7AAD. Histograms of absolute frequencies of CTV dilution for total live and dead singlet cells (dashed gray), live cells with (fi

Supportive validation

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Experimental details: Fig. 4 Bach2 is intrinsically required for pTreg cell differentiation. a Flow cytometry plots and quantification showing GATA3, RORgammat and Helios expression by Treg cells from the colonic lamina propria of Bach2 fl/fl Cd4 Cre and control mice. b Flow cytometry plots showing Foxp3 expression by wildtype and Bach2 -/- CD4 T cells after three days in Treg cell-inducing culture conditions, and frequency of Foxp3-expressing cells (right). c Histograms showing Blimp1-GFP expression by Blimp1 GFP and Bach2 -/- Blimp1 GFP CD4 T cells after culture in Treg cell-inducing conditions. Dashed line indicates background fluorescence levels in non-reporter cells. d Flow cytometry plots showing Foxp3 and IL-10 expression by wildtype and Bach2 -/- CD4 T cells after Treg cell-inducing culture, and quantification (right). e Flow cytometry plots showing Foxp3 expression by wildtype, Bach2 -/- , Blimp1 fl/fl Lck Cre and Bach2 -/- Blimp1 fl/fl Lck Cre after three days culture in Treg cell-inducing conditions, and frequency of Foxp3 and IL-10 expressing cells (below). Flow cytometry plots are representative, data pooled from ( a ) or representative of two ( c - e ) or six ( b ) independent experiments. Statistical significance tested using two-way ANOVA with Tukey's test ( e ). Otherwise, significance was tested using the unpaired Student's t- test. Error bars denote mean +- S.D. Source data are provided as a Source Data file.